Locomotion is a fundamental behavior in vertebrates, in which motor neurons (MNs) play a central role by establishing precise connections between the central nervous system and the periphery. Hox transcription factors are essential in defining the cell type specific molecular identity of MNs, which is achieved through temporal and spatial regulation of Hox gene expression. Polycomb group (PcG) proteins were initially discovered in Drosophila embryo as regulators of Hox genes, whose expression defines body structures and appendage position, and are conserved in the vertebrate system. PcG proteins assemble into two multimeric complexes, PRC1 and 2, and maintain gene repression through chromatin modifications. However, specific roles of PRC1 in neuronal fate determination are poorly understood. We have recently found that a PRC1 component, Bmi1/PCGF4, plays an essential role in motor neuron (MN) subtype differentiation, by creating gradient of PRC1 activity along rostrocaudal axis of spinal cord and defining the rostral boundary of Hox gene expression in MN columns at the post-mitotic level. However, the specific mechanisms of PRC1 action remain unanswered. This proposal attempts to answer the following questions: (1) Which subunits of PRC1- Bmi1/PCGF4 complex contribute to the PRC1 activity gradient? (2) Do additional PRC1 complexes containing PCGF1,2,3,5 or 6 play any role in controlling Hox expression during MN development? To address these questions I will first define the expression profiles of PRC1 subunits by in situ hybridization and immunofluorescence and determine their localization to column defining-Hox loci by chromatin immunoprecipitation. In addition I will address the function of PRC1 complexes in establishing Hox expression pattern by depleting and overexpressing its components in developing chick embryo by in ovo electroporation. The overall goal of this project is (1) to dissect the mechanism in which columnar-defining Hox gene expression is maintained in the developing spinal cord, and (2) to extend our general understanding of the functions and mechanisms of Polycomb repression in the developing embryo, using MN specification as a model system.